1. Field of the Invention
The present invention relates to a method for obtaining crystals of a basic amino acid hydrochloride from a basic amino acid fermentation broth or an enzyme reaction solution which enzyme reaction has been catalyzed with viable cells of a basic amino acid-producing microorganism, wherein the broth or the solution contains sulfate ions.
2. Brief Description of the Related Art
Basic amino acid fermentation broths and enzyme reaction solutions typically contain sulfate ions which are derived from the ammonium sulfate which is present as a nitrogen source in either the fermentation media or substrate solutions for the enzyme reaction.
Conventionally, in order to obtain crystals of a basic amino acid hydrochloride from such a basic amino acid fermentation broth or an enzyme reaction solution, which broth or solution contains sulfate ions, at a high purity, first, the basic amino acid fermentation broth or the enzyme reaction solution is passed through an ammonium-type cation exchange resin column. The basic amino acids are adsorbed onto the resin, while the sulfate ions are removed in the form of an ammonium sulfate solution together with the exchanged or desorbed ammonium ions. Thereafter, the ion exchange resin on which the basic amino acids have been adsorbed is washed with an ammonia solution to elute the basic amino acids, and the basic amino acids are then concentrated as the free form in the eluate. Finally, the free basic amino acid that is obtained is neutralized with hydrochloric acid, whereby basic amino acid hydrochloride crystals are formed and obtained from the mother liquor.
However, this method can be problematic in the following respects. Namely, (1) the eluted ammonium sulfate solution must be concentrated using an enormous amount of vapor so that the byproduct of ammonium sulfate can be recycled, and (2) a large amount of waste water is discharged when the resin is washed.
Other methods for obtaining a basic amino acid include, for example, a method wherein a lysine fermentation broth is supplied with a potassium hydroxide solution to crystallize the lysine base (free form) (European Patent Publication No. 0534865), and a method wherein a lysine fermentation broth is filtered through activated carbon to remove the cells of the chosen lysine-producing microorganism, then the resulting filtrate is mixed with a calcium hydroxide solution which serves to deposit and remove the calcium sulfate which is generated, and finally the remainder is concentrated to remove the ammonia, and the lysine base is obtained (Russian Patent Publication No. 183581). Then, the lysine base is mixed with hydrochloric acid, whereby lysine hydrochloride crystals are obtained.
However, the methods described above wherein an expensive metal hydroxide and hydrochloric acid are used as auxiliary materials and the resulting by-product metal sulfate is very cheap compared with the metal per se, result in a manufacturing procedure wherein the costs for the auxiliary materials are high. Furthermore, after mixing with a metal hydroxide, the solution is concentrated to a pre-determined lysine concentration by using heat at a high pH, which results in degradation of the lysine.
Moreover, these methods involve a suspension which contains metal sulfate crystals and a basic amino acid. However, the basic amino acid is in the free base form in the suspension, and therefore, the suspension is viscous and it is difficult to separate the metal sulfate crystals from the basic amino acid. As a result, a large amount of the amino acid adheres to the metal sulfate crystals to be discharged, resulting in a lower recovery rate of the amino acid of interest.
Exemplary methods of the present invention involve a suspension which contains the amino acid in the hydrochloride form and thus is not very viscous, which allows for easy separation of the metal sulfate crystals from the amino acid moiety and increases the recovery rate of the amino acid.
In addition, other problems exist, such as the metal hydroxide in aqueous solution is usually added at a concentration of not more than 50% which results in a decrease in the amino acid concentration in the system. This raises the cost for producing the vapor. 100% of metal hydroxide powder may be added to the system, but to do so is dangerous and difficult; and in addition, the separate required steps of adding the metal hydroxide and the hydrochloric acid complicates the production operation.
Previously, a metal chloride was not thought to react with a basic amino acid sulfate in a basic amino acid fermentation broth because it was believed that no anion exchange reaction occurs between the basic amino acid sulfate and the metal chloride. Namely, it was thought to be impossible that an equilibrium reaction would occur between the salts because the system consists of the basic amino acid sulfate in the solution phase and the metal chloride in the solid phase. It was believed that, in such a solid-liquid system, even if the metal chloride is added as a solid, the metal chloride would remain as a solid, while the dissolved basic amino acid sulfate would stay dissolved.
The amount of basic amino acids produced is not limited as long as they are generated by fermentation or by enzymatic methods using microbial cells as a catalyst. These amino acids include, for example, arginine, histidine and lysine. The form of the amino acids is not limited, but an L-form is one example.
Exemplary microbes according to the present invention include those which are able to produce the target amino acid or those which are able to catalyze the reaction to produce the target amino acid from substrates. The former are used in fermentation methods while the latter are used in enzymatic methods. As microbes, any bacteria, yeasts, filamentous bacteria and the like may be used, bacteria being one example. Bacteria may either be Gram-negative or Gram-positive. The microbe may be used alone or in combination with one or more other microbes.
L-lysine producing bacteria and methods for breeding the same are known, including those disclosed in, for example, WO 95/23864, WO 96/17930, WO 2005/010175, Japanese Patent Application Laid-Open (Kokai) Sho No. 56-18596, U.S. Pat. No. 4,346,170, and Japanese Patent Application Laid-Open (Kokai) No. 2000-189180. L-arginine producing bacteria and methods for breeding the same are also known, and include those disclosed in, for example, United States Patent Application Publication No. 2002/058315A1, Russian Patent Application No. 2001112869, EP 1170358A1 and EP 1170361A1. L-histidine producing bacteria and methods for breeding the same are also know, and include those disclosed in, for example, Russian Patent Nos. 2003677 and 2119536, U.S. Pat. Nos. 4,388,405, 6,344,347 and 6,258,554, Russian Patent Nos. 2003677 and 2119536, Japanese Patent Application Laid-Open (Kokai) Sho No. 56-005099 and EP1016710A. The same is the case with known L-ornithine producing bacteria and methods for breeding the same.